The cAMP-dependent protein kinase (PKA) is thought to be maintained at specific sites through out the cell by interaction of its regulatory subunit (R) with anchoring proteins. We propose that PKA anchoring influences which substrate proteins become phosphorylated by individual kinase pools. The long term goals of this laboratory are to determine the role of PKA localization in regulating cAMP mediated responses. Toward this goal, each specific aim of this proposal will characterize structural aspects o the R subunit/ anchoring protein interactions. Previous work in our laboratory has shown that RII-anchoring proteins bind to the same or overlapping sites on the type II regulatory subunit and imply that each RII-anchoring protein may contain a common structural motif which represents the RII-binding site. Our analysis of four RII-anchoring proteins suggests that RII-binding sequences consist of 14 residues and can be divided into two distinct groups, which may represent isoform specific binding motifs. Specific aim 1 of this proposal will attempt to define isoform specific RII-binding sites by screening a "random peptide library" using recombinant RIlalpha or RIIbeta as probes. We have recently synthesized a peptide analog derived from Ht 31 an RII anchoring protein which binds RII with nanomolar affinity. Specific aim 3 will use this peptide in chemical cross-linking studies to determine: a) the stoichiometry of RIIalpha/Ht 31 peptide interaction, and b) which amino-acids on RIIalpha bind to the anchoring protein. PKA localization may be regulated by protein kinase catalyzed phosphorylation of the R subunit. Soluble RII, isolated from various tissues, contains up to 2 molecules of phosphate per protomer incorporated at sites which lie within the anchoring protein receptor site. Specific aim 4 is to determine if phosphorylation by these protein kinases alters RIIalpha affinity for Ht 31 peptides. Equilibrium dialysis will be used to compare the binding affinities of the phosphorylated and unphosphorylated RIIalpha. Recent evidence from our laboratory suggests that the type I PKA may be also be specifically localized through interaction of the type I R subunit with anchoring proteins. This is an important finding as it suggests that both PKA subtypes may be specifically localized in selected tissues. Therefore, specific aim 2 will use recombinant RIalpha as a probe to screen a pituitary cDNA expression library to isolate clones to this RI-anchoring protein.